K ؉ channels operate in the plasma membrane and in membranes of organelles including mitochondria. The mechanisms and topogenic information for their differential synthesis and targeting is unknown. This article describes 2 similar viral K ؉ channels that are differentially sorted; one protein (Kesv) is imported by the Tom complex into the mitochondria, the other (Kcv) to the plasma membrane. By creating chimeras we discovered that mitochondrial sorting of Kesv depends on a hierarchical combination of N-and C-terminal signals. Crucial is the length of the second transmembrane domain; extending its C terminus by >2 hydrophobic amino acids redirects Kesv from the mitochondrial to the plasma membrane. Activity of Kesv in the plasma membrane is detected electrically or by yeast rescue assays only after this shift in sorting. Hence only minor structural alterations in a transmembrane domain are sufficient to switch sorting of a K ؉ channel between the plasma membrane and mitochondria.algal viruses ͉ dual targeting ͉ Kϩ channel sorting ͉ PBCV-1 ͉ Esv-1

A computational model for the open state of the short viral Kcv potassium channel was created and tested based on homology modeling and extensive molecular-dynamics simulation in a membrane environment. Particular attention was paid to the structure of the highly flexible N-terminal region and to the protonation state of membrane-exposed lysine residues. Data from various experimental sources, NMR spectroscopy, and electrophysiology, as well as results from three-dimensional reference interaction site model integral equation theory were taken into account to select the most reasonable model among possible variants. The final model exhibits spontaneous ion transitions across the complete pore, with and without application of an external field. The nonequilibrium transport events could be induced reproducibly without abnormally large driving potential and without the need to place ions artificially at certain key positions along the transition path. The transport mechanism through the filter region corresponds to the classic view of single-file motion, which in our case is coupled to frequent exchange of ions between the innermost filter position and the cavity.

Some algal viruses contain genes that encode proteins with the hallmarks of K(+) channels. One feature of these proteins is that they are less than 100 amino acids in size, which make them truly minimal for a K(+) channel protein. That is, they consist of only the pore module present in more complex K(+) channels. The combination of miniature size and the functional robustness of the viral K(+) channels make them ideal model systems for studying how K(+) channels work. Here we summarize recent structure/function correlates from these channels, which provide insight into functional properties such as gating, pharmacology and sorting in cells.

The viral potassium channel Kcv comprises only 94 amino acids, which represent the pore module of more complex K(+) channels. As for Kir-type channels, Kcv also has a short N-terminal helix exposed to the cytoplasm, upstream of the first transmembrane domain. Here we show that this helix is relevant for Kcv function. The presence of charged amino acids, which form dynamic inter- and intra-subunit salt bridges is crucial. Electrophysiological measurements, yeast rescue experiments and molecular dynamics simulations show that mutants in which the critical salt bridge formation is impaired have no or reduced channel activity. We conclude that these salt bridges destabilise the complexation of K(+) ions by negative charges on the inner transmembrane domain at the entrance into the cavity. This feature facilitates a continuous and coordinated transfer of ions between the cavity and the cytoplasm for channels without the canonical bundle crossing.

Transmembrane domains (TMDs) are often flanked by Lys or Arg because they keep their aliphatic parts in the bilayer and their charged groups in the polar interface. Here we examine the relevance of this so-called "snorkeling" of a cationic amino acid, which is conserved in the outer TMD of small viral K + channels. Experimentally, snorkeling activity is not mandatory for Kcv PBCV-1 because K29 can be replaced by most of the natural amino acids without any corruption of function. Two similar channels, Kcv ATCV-1 and Kcv MT325 , lack a cytosolic N-terminus, and neutralization of their equivalent cationic amino acids inhibits their function. To understand the variable importance of the cationic amino acids, we reanalyzed molecular dynamics simulations of Kcv PBCV-1 and N-terminally truncated mutants; the truncated mutants mimic Kcv ATCV-1 and Kcv MT325 . Structures were analyzed with respect to membrane positioning in relation to the orientation of K29. The results indicate that the architecture of the protein (including the selectivity filter) is only weakly dependent on TMD length and protonation of K29. The penetration depth of Lys in a given protonation state is independent of the TMD architecture, which leads to a distortion of shorter proteins. The data imply that snorkeling can be important for K + channels; however, its significance depends on the architecture of the entire TMD. The observation that the most severe N-terminal truncation causes the outer TMD to move toward the cytosolic side suggests that snorkeling becomes more relevant if TMDs are not stabilized in the membrane by other domains.